(1) Field of the Invention
The present invention relates to a method and test kit for detecting the presence of a detectable or first antibody in a sample using a gold-labeled second antibody to the first antibody and microspheres coated with an antigen reactive with the first antibody in a sandwich type assay method. In particular, the present invention relates to a method wherein an electron microscope is preferably used for the detection of the number of gold particles in the antigen-antibody complex on the microsphere which is related to the detectable antibody in the sample. The method is qualitative and can be quantitative.
(2) Prior Art
The detection of specific antibody in sera continues to be one of the surest ways of diagnosing autoimmune diseases and infections by various types of pathogenic agents. Solid phase immuno-assays based on enzyme-linked immunosorbent assays-ELISA (Engvall, E. and Perlman, P., Immunochemistry 8, 871 (1971) are among the most widely used techniques for detection of specific antibodies (Butler, J. E., et al. J. Immunol. Methods 20, 365 (1971); Johannsson, A., et al., J. Immunol. Methods 87, 7 (1986); Logtenberg, T., et al., Immunol. Lett. 9, 343 (1985); Sedgwich, J. D., et al., J. Immunol. Methods 87, 37 (1986); Kemeny, D. M. et al., Immunol. Today 7, 67 (1986); and Ljunggren, K., et al., J. Immunol. Methods 104 (1987)). Indeed, several improvements have been made to ELISA such that these techniques are fast replacing many isotopic (radiolabel) immunoassays (Kemeny, D. M., et al., Immunol. Today 7, 67 (1986)).
U.S. Pat. Nos. 4,420,558 and 4,446,238 to DeMey et al show the use of gold-labeled antibodies for detecting subtypes of blood cells or for localizing antigens in blood tissues. These methods are used for antigen detection rather than antibody detection. Examples of other related art in the use of gold-labeled antibodies is set forth in J. Immunol. Methods 106, 217-223 (1988) 74, 353-360 (1984); and GB No. 8331514 (831125).
The technique of coating spheres with specific antibodies for detection of cell surface and other antigens has been described by other investigators (Stiffler-Rosenberg, G. and Fey, H., J. Clin. Microbiology, 8, 473 (1978); Rembaum, A., and Dreyer, W. J., Science 208, 364 (1980); and Bourel, D., Rolland, A., LeVerge, R., and Genetet, B., J. Immunol. Methods 106, 161 (1988)). The problem is the visualization of the results.